The present application relates to a fine particle measurement apparatus and an optical axis calibration method. More particularly, the disclosure relates to a fine particle measurement apparatus capable of automatically adjusting a positional relationship between a sample flow, where fine particles pass through, and a condensing spot of the light irradiated to the sample flow.
In the related art, a fine particle measurement apparatus is widely used, in which light (laser) is irradiated to an inner side of a flow cell or fine particles flowing through a flow path formed on a microchip, dispersed light from fine particles, fluorescent light generated from the fine particles or fluorescent substances labeled onto the fine particles is detected to measure optical properties of the fine particles. In such a fine particle measurement apparatus, a population (group) satisfying a predetermined condition as a result of the measurement of optical properties is often separately retrieved from the fine particles. In such an apparatus, a device capable of measuring the optical properties of, particularly, cells as the fine particles, or separately retrieving a cell group satisfying a predetermined condition is called a flow cytometer or a cell sorter.
For example, Japanese Unexamined Patent Application Publication No. 2007-46947 discloses “a flow cytometer having a plurality of light sources for irradiating a plurality of excitation light beams having different wavelengths in a predetermined cycle with different phases and an optical guide member for guiding a plurality of excitation light beams into the same incident optical path to condense them onto a dyed particle.” The flow cytometer includes a plurality of light sources for irradiating a plurality of excitation light beams having different wavelengths, an optical guide member for guiding a plurality of the excitation light beams into the same incident optical path to condense them onto a dyed particle, and a plurality of fluorescence detectors for detecting fluorescent light generated from the particles excited by each of excitation light beams to output fluorescent signals (refer to Claims 1 and 3 and FIGS. 1 and 3 of Japanese Unexamined Patent Application Publication No. 2007-46947).
In the fine particle measurement apparatus of the related art, as shown in FIG. 8, the laser L is condensed from a direction approximately perpendicular to the sample flow S using the condensing lens 103. The fine particles P pass through the sample flow S to cross the spot of the condensed laser L. In this case, the intensity distribution of the laser spot has become a Gaussian distribution in which the intensity distribution is strong in the center of the spot and significantly decays at the periphery. FIG. 9 illustrates an exemplary intensity distribution of the laser spot in the fine particle measurement apparatus of the related art. For this reason, when the flow-sending position of the fine particles P within the sample flow S matches with the center position of the laser spot, the effective intensity of the laser irradiated to the fine particles P is maximized, and the obtained signal intensity is also maximized.
The operation of matching the flow-sending position of fine particle within the sample flow with the center position of the laser spot is generally called an “optical axis calibration.” The optical axis calibration is carried out by flowing calibration micro beads and calibrating the position or focus of the condensing lens or centering the light source while histogram data is referenced to optimize the relative positions of the laser, the sample flow, the detector, and the like. Japanese Unexamined Patent Application Publication Nos. 11-83724 and 9-196916 also disclose calibration micro beads used in the optical axis calibration.